The proposed studies seek to determine the molecular events involved in the tissue-specific, regulated expression of the prolactin gene. Attention will be focused on specific DNA sequences which have been shown to be important for prolactin gene expression and on the transcription factors which interact with these sequences. The specific aims include: 1) Analysis of the functional role of an element in the proximal 5' flanking region of the gene which has similarity to known cAMP-responsive elements. These studies will involve efforts to purify, characterize and clone the factors which interact with this site. 2) Investigate mechanisms which may account for functional cooperativity between different DNA elements within the prolactin gene. This would include analysis of possible interactions of Pit-1 with the general transcription factors, TFIID and TFIIB. Other studies would examine interactions between the distal and proximal regions of the prolactin gene which are required for an estrogen response. 3) Examine possible interactions between Pit-1 and the estrogen receptor. Preliminary studies have shown that Pit-1 as well as the estrogen receptor are required for an estrogen response of the prolactin gene. This suggests that the non-palindromic estrogen receptor binding site of the prolactin gene functions somewhat differently than the palindromic receptor binding sites found in some other genes. As the estrogen receptor binding site is immediately adjacent to a Pit-1 binding site, a role for physical interactions with Pit-1 seems reasonable. Possible interactions between Pit-1 and the estrogen receptor will be examined in solution as well as for DNA binding. 4) The functional significance of Pit-1 phosphorylation will be examined. This will involve transfection studies utilizing Pit-1 expression vectors containing mutations which prevent phosphorylation at specific sites. The possible role of Pit-1 phosphorylation in DNA bending will also be examined. 5) Biochemical studies will be used to examine Pit-1 structural features. This will include chemical modification, and protease sensitivity studies as well as x-ray crystallographic approaches. 6) Utilize a yeast expression system for a molecular genetic analysis of important Pit-1 structural features. The yeast system will be used to identity point mutations that alter the ability of Pit-1 to activate transcription and to search for other transcription factors which are important for expression of the prolactin gene. Overall these studies aim to increase understanding of the structure, function and interactions of transcription factors which are important for regulating expression of the prolactin gene.